This project will investigate the molecular mechanisms that ensure that each egg is fertilized by only one sperm, thus allowing normal embryonic development to occur. In many animal species, one essential component of polyspermy prevention is an electrical depolarization of the egg membrane in response to fusion of the first sperm, which prevents the fusion of additional sperm. The sensor that detects the voltage change in the egg is known to be located in the sperm, but has not been identified on a molecular level. The proposed studies will investigate this question, focusing on a recently discovered voltage-activated phosphatidylinositol lipid phosphatase that is expressed in sperm. A close correlation between the voltage that activates the phosphatase and the voltage that inhibits sperm-egg fusion supports this hypothesis. Specific aim one will investigate the localization of the voltage-sensitive phosphatase (VSP) in the sperm membrane. Localization in the head region, where sperm-egg fusion occurs, would support a role for the VSP in the regulation of this process. Alternatively, localization in the tail region would support its role in the regulation of sperm motility. Specific aim two will investigate the function of the VSP, using a transgenic frog whose sperm express a point-mutated form of the protein in which the phosphatase activates at a more negative voltage. The direction of these functional experiments will be determined by the results of aim one. If the VSP is localized in the sperm head, transgenic sperm will be applied to voltage-clamped eggs to test whether the presence of the modified VSP shifts the voltage that inhibits sperm-egg fusion. Alternatively, if the VSP is localized in the sperm tail, the effect of the modified VSP on sperm motility will be investigated. By taking advantage of a unique biological system in which sperm-egg fusion is voltage dependent, these studies will provide insights into the possible function of phosphatidylinositol lipids in regulating the fusion process, and will test the role of a voltage-sensitive phosphatase in this and other physiological regulatory mechanisms in sperm. The findings will contribute to understanding of the biology of fertilization, and will provide a basis for future advances in reproductive medicine. PUBLIC HEALTH RELEVANCE: This project will investigate the molecular mechanisms that ensure that each egg is fertilized by only one sperm, thus allowing normal embryonic development to occur. The findings will contribute to understanding of the biology of fertilization, and will provide a basis for future advances in reproductive medicine.